This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The major human bacterial pathogens Streptococcus pyogenes and Staphylococcus aureus have evolved the systems to efficiently acquire heme from host hemoproteins as a preferred source of essential iron. Thus, these systems provide several potential therapeutic targets, and the mechanistic study on these systems will provide clinically relevant antibiotic strategies to inhibit the heme acquisition process mediated by these systems for treating S. pyogenes and S. aureus infections. Translational studies to develop the antibiotic strategies need to first understand and establish the molecular mechanism of the heme acquisition process and its structural basis. Thus, Dr. Lei's lab has designed an in vitro strategy to elucidate the pathway of heme acquisition in these pathogens. These studies physically demonstrated heme transfer from one protein to another and used kinetic analysis to determine whether the heme transfer is directional. Dr. Lei's previous results indicated the S. pyogenes pathway of heme transfer from hemoglobin to Shr, then to Shp, and finally to HtsA. Dr. Lei has now discovered that for the S. aureus system: (1) hemoglobin directly transfers heme to apo-IsdB;(2) holo-IsdB directly transfers heme to apo-IsdA and apo-IsdC, but not to apo-IsdE;(3) apo-IsdE directly acquires heme from holo-IsdC, but not from holo-IsdB and holo-IsdA;and (4) IsdB and IsdC enhance heme transfer from hemoglobin to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. These results establish a S. aureus heme transfer pathway of hemoglobin IsdB IsdA IsdC IsdE. These results greatly enhance our understanding of the mechanisms of heme acquisition in S. pyogenes and S. aureus and may serve as a general model for heme acquisition in many other Gram-positive pathogens. Understanding of how these bacteria utilize heme to survive in humans may provide clues on how to block the heme transport for developing new therapeutics to combat these harmful pathogens.